Recent studies showed that a cellular protein known as HM1.24, CD317, BST-2, or tetherin (hereafter referred to as BST-2/tetherin) inhibits HIV-1 particle release by tethering newly assembled virus particles to the plasma membrane (PM) of virus-producing cells. However, the mechanism by which BST-2/tetherin is incorporated into assembling virus particles remains to be determined. Notably, BST-2/tetherin can inhibit release of a wide variety of enveloped viruses including retroviruses, filoviruses, herpesviruses, rhabdoviruses, paramyxoviruses, and arenaviruses. Therefore, determining the mechanism that recruits BST-2/tetherin to HIV-1 assembly sites will not only advance our understanding of the biology of HIV that causes AIDS but also will help us develop antiviral strategies targeting a broad range of enveloped viruses. Notably, an artificial protein that shares key structural features with BST-2/tetherin with no sequence homology was capable of blocking HIV-1 release. Therefore, association of BST-2/tetherin to nascent virus particles is likely dependent on a common cellular structure rather than specific viral or cellular proteins. The PM is now thought to consist of multiple microdomains rather than a uniform lipid bilayer with proteins embedded in a random manner. These microdomains contain specific sets of lipids and proteins and may have various lifetimes, sizes, and dynamics. Among them, lipid rafts are well known to associate with HIV-1 assembly. Notably, BST-2/tetherin is modified at its C terminus with a GPI anchor, which is known to target proteins to lipid rafts. Together with the notion that many enveloped viruses associate with lipid rafts or other microdomains, it is conceivable that association of BST-2/tetherin with lipid rafts may promote incorporation of this protein to assembling particles. However, it is also possible that other membrane-associated structures common to BST-2/tetherin-susceptible viruses, such as membrane curvature, membranous neck/stalk, or ESCRT (endosomal sorting complex required for transport) proteins, play a role in BST-2/tetherin recruitment. Toward better understanding of the antiviral activity of BST-2/tetherin, in this proposal, we plan to test the following hypothesis: BST-2/tetherin associates with specific PM structures that are recruited to virus assembly sites. To test this hypothesis, in Aim 1, the order of events with regard to incorporation of rafts, ESCRTs, and BST-2/tetherin during virus assembly will be determined. Using combination of genetic approaches with advanced microscopy techniques, we plan to identify the assembly step at which BST-2/tetherin are recruited to assembly sites. Experiments in Aim 2 are designed to determine whether lipid rafts and ESCRTs are essential for BST- 2/tetherin function and its recruitment to virus assembly sites. Altogether, information gained in experiments proposed in this application will likely help us elucidate the mechanism that initiates BST-2/tetherin-mediated restriction of HIV-1 release.